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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Rev. Int. Contam. Ambie. 30 (1) 51-63, 2014
REMOVAL OF METHYL PARATHION AND COUMAPHOS PESTICIDES BY A BACTERIAL
CONSORTIUM IMMOBILIZED IN
Luffa cylindrica
David Antonio MORENO-MEDINA, Enrique SÁNCHEZ-SALINAS y
Ma. Laura ORTIZ-HERNÁNDEZ*
Laboratorio de Investigaciones Ambientales, Centro de Investigación en Biotecnología, Universidad Autónoma
del Estado de Morelos. Av. Universidad 1001, Col. Chamilpa, C.P. 62209, Cuernavaca, Morelos, México
*Corresponding author; ortizhl@uaem.mx
(Recibido febrero 2013, aceptado octubre 2013)
Key words: pesticides, biodegradation, immobilized cells, bioflm,
Luffa
cylindrica
ABSTRACT
The constant application of pesticides has led to environmental and health problems in
many regions of the world, as well as the storage of large amounts of pesticide waste and
obsolete pesticides. For this reason, it is necessary to develop strategies for the disposal
of pesticides that are ecofriendly and economically viable. Different treatment options
exist, but in recent years, the application of biological systems has gained the greatest
acceptance, because it promises the degradation and detoxifcation oF pesticides without
harming the environment or human health. In this study, we used a bacterial consortium
that was isolated from agricultural soils to degrade a mixture of the organophosphate
pesticides methyl parathion (MP) and coumaphos (COU). The eFfciency oF removal
was evaluated using mineral salt medium supplemented with glucose, and the bacterial
consortium was cultivated as free cells and immobilized on
Luffa cylindrica
fbers. To
improve the structure oF the fbrous network and to achieve greater retention oF micro
-
organisms, removal was also tested prior to fbers treatment with sodium hydroxide
(NaOH). The results indicate that the microorganisms used had better growth as free
cells. A removal of 54.88 % and 62 % for MP and COU, respectively was observed
using the Free cells; but when the cells were immobilized on looFa sponge fbers, the
removal was increased to 98 and 100 % of those pesticides. This pesticide removal was
the result of a combined effect among the activity of the microorganisms, the adhesion
to the bacterial cells and the adsorption on the support material.
We observed a strong
and Fast adsorption on the looFa sponge fber, since removal obtained only with looFa
fber, did not present signifcant diFFerence with immobilized microorganisms. ±urther
studies are needed to understand the processes that occur with adsorbed pesticides and
whether there was subsequent desorption and degradation.
Palabras clave: plaguicidas, biodegradacion, células inmovilizadas, biopelícula,
Luffa cylindrica
RESUMEN
La aplicación constante de plaguicidas ha traído como resultado problemas ambienta-
les y de salud pública en muchas regiones del mundo, además del almacenamiento de
grandes cantidades de residuos y de plaguicidas obsoletos. Por esta razón, es necesario
desarrollar estrategias económica y ambientalmente viables para el tratamiento y dis-
D.A. Moreno Medina
et al.
52
posición fnal de los plaguicidas. Existen diFerentes opciones, aunque recientemente la
aplicación de sistemas biológicos ha ganado gran aceptación debido a la posibilidad de
lograr la degradación y detoxifcación de los plaguicidas sin causar daños al ambiente o
a la salud. En este estudio se utilizó un consorcio bacteriano aislado de suelos agrícolas
para degradar una mezcla de paratión metílico (PM) y coumafos (COU), dos plaguicidas
organoFosForados. Se evaluó la efciencia de remoción del consorcio utilizando un medio
de sales minerales con adición de glucosa, además de que el consorcio fue cultivado
en suspensión e inmovilizado en fbras de
Luffa cylindrica
. Para mejorar la estructura
de la red fbrosa de la planta y lograr una mayor retención de los microorganismos, la
remoción se probó con y sin un pretratamiento con hidróxido de sodio. Los resultados
indican que los microrganismos utilizados presentaron mejor crecimiento en suspensión
y se observó una remoción del 54.88 % y 62 % de PM y COU, respectivamente; sin
embargo, cuando las células Fueron inmovilizadas en las fbras de
Luffa cylindrica
, la
remoción se incrementó a 98 % y 100 % respectivamente. Esta remoción fue el resultado
de un efecto combinado entre la actividad de los microorganismos, la adhesión a las
células bacterianas y la adsorción al material de soporte. Se observó una rápida y fuerte
adsorción sobre las fbras del material soporte, ya que la remoción obtenida sólo con la
fbra, no presentó diFerencias signifcativas con los microorganismos inmovilizados. Se
requieren mayores estudios para entender los procesos que ocurren con los plaguicidas
adsorbidos y su posible desorción y degradación posterior.
INTRODUCTION
Increasing population, growing demand for
food, agricultural mechanization and the need to
control a variety of new pests have resulted in an
increased production and consumption of pesticides.
In addition to increasing agricultural production,
the benefts oF pesticides For controlling pests and
disease vectors are undeniable. However, every
year, pesticides must be applied at higher doses, and
this intensive use of pesticides has irreversible ad-
verse effects on the environment and human health
(Soares
et al
. 2003, Recena
et al
. 2006). A pesticide
is any substance or mixture of substances intended
to prevent, destroy or control pests, which cause
damage or otherwise interfere in the production,
processing, storage, transportation or marketing oF
food, agricultural products and wood. Vectors or
intermediate hosts of human and animal diseases
that can be supplied to animals in order to fght
against any organism inside or on their bodies are
also considered pesticides (Athia 2006).
It is estimated that approximately 85 % of pes-
ticides used worldwide are used for agriculture
(Cervantes 2010). In 2007, 2.36 million tons of active
pesticides were consumed for agricultural purposes
worldwide (USEPA 2011). The continuous applica-
tion of pesticides has increased their concentration
in soil and water, which leads to their entry into the
food chain (Ortiz-Hernández
et al.
2011). Disper-
sion mechanisms have also increased the level of
environmental risk For the occupationally exposed
population and the inhabitants of surrounding vil-
lages. On the other hand, liquid and solid waste and
obsolete products that are stored or disposed of in
an inappropriate manner have resulted in signifcant
environmental liabilities, which in most cases are
not reported to the appropriate authority (Sánchez-
Salinas and Ortiz-Hernández 2011).
Pesticides of different chemical structures are
toxic and persistent in the environment. Currently,
organophosphates (OP) are used worldwide as
pesticides or chemical warfare agents because of
their high toxicity toward insects, mammals and
other animals (Theriot and Grunden 2011). These
compounds have a basic structure that consists of
ester or thiol derivatives of phosphoric, phosphonic
or phosphoramidic acids (Vilanova and Sorgob
1999, Ortiz-Hernández and Sánchez-Salinas 2010).
Their mechanism of action involves the irreversible
inhibition oF acetylcholinesterase, a key enzyme oF
the central nervous system, and thus, they affect non-
target organisms (Singh and Walker 2006). Like all
pesticides, intensive use of OP pesticides can lead
to their accumulation in the environment and may
affect ecosystems and human health.
To mitigate the problem of contamination by OPs,
treatments have been developed to detoxify and/or
degrade these pesticides through physical, chemical
and/or biological processes. In biological processes,
biological systems (whole cells or isolated enzymes)
are used to catalyze chemical reactions that transform
the pesticide into simpler and less toxic compounds
or, better yet, mineralize them into molecules.
REMOVAL OF PESTICIDES BY AN IMMOBILIZED BACTERIAL CONSORTIUM
53
Biological methods are gaining interest due to
their simplicity, high efFciency and cost effective
-
ness compared to other methods (Chandran and Das
2011). Biodegradation of OP pesticides provides a
cheap and efFcient solution for their Fnal disposal or
for the treatment of agricultural soils, contaminated
water or polluted ecosystems. To date, a number of
different microorganisms have been identiFed, and
the enzymes involved in OP degradation have been
studied (Singh 2009, Ortiz-Hernández
et al
. 2011,).
In order to optimize biological treatment, different
strategies have been developed. Among them, cell
immobilization has been a successful approach that
ensures that the catalytic activity of the biological
processes is maintained for longer periods (Manohar
et al
. 2001, Cheng
et al
. 2003, Yáñez-Ocampo
et al
.
2009). Immobilization consists in restricting cell
mobility within a deFned space of a material with
particular characteristics.
There are two types of processes for cell immo-
bilization: those based on physical retention (entrap-
ment and inclusion membrane) and those based on
chemical bonds, such as bioFlm formation (Kennedy
and Cabral 1983). In cell immobilization methods,
various inorganic (clays, silicates, glass and ceram-
ics) and organic (cellulose, starch, dextran, agarose,
alginate, chitin, collagen, keratin, etc.) supports are
employed (Arroyo 1998).
The most widely studied method for the practi-
cal application of immobilized cell techniques is
entrapping cells in polymer gels, such as alginate
and carrageenan. However, this method has been
limited by problems with gel stability and by the
mass transfer limitations of gel beads. Entrapment
in natural polymeric gels has become the preferred
technique for the immobilization of cells, due to the
toxicity problems associated with the synthesis of
polymeric materials (Lusta
et al
. 1990). However,
biodegradable natural supports have been used more
frequently in the treatment of sewage contamination
(Garzón-Jiménez 2009).
Several reports indicate that a variety of materi-
als provide the necessary features to immobilize
microorganisms (Jin
et al
. 1998, Iqbal and Saeed
2006, Barragán
et al
. 2007, May-Esquivel
et al
. 2008,
Garzón-Jiménez 2009). Pattanasupong
et al
. (2004)
reported on the use of various plant Fbers as supports
for immobilizing a bacterial consortium to degrade
xenobiotics. The use of the natural structural materi-
als, such as the petiolar felt-sheath of palm, for cell
entrapment has added another dimension to a variety
of immobilization matrices (Iqbal and Saeed 2006,
Iqbal and Edyvean 2007). The advantages of such
biostructures are their reusability, no toxicity, me-
chanical strength and open spaces within the matrix
for growing cells, which avoids rupture and diffusion
problems (Akhtar
et al
. 2004). These Fndings led
us to search diverse plant sources for other types of
biomaterials that may be used for cell entrapment.
Among these materials, the Fbers of
Luffa cylin-
drica
L. (sponge loofa) have been used as a natural
support to immobilize various microorganisms.
Among them
Chlorella sorokiniana, Porphyridium
cruentrum, Penicillium cyrlopium,
and
Funalia tro-
gii
, for nickel and cadmium II treatment, as well as
for use in dyes and chlorinated substances (Akhtar
et al
. 2004, Mazmanci and Ünyayar 2005, Alluri
et al
. 2007). Furthermore, the use of
Luffa
sp. to
immobilize fungal biomass for metal biosorption
processes was previously reported by Iqbal and
Edyvean (2007).
Loofa
(Luffa cylindrica)
grows well in both
tropical and subtropical climates, and the sponges are
produced in large quantities in México, where they
are currently used for bathing and dish washing. The
sponges are light, cylindrical in shape and made up
of interconnecting voids within an open network of
matrix support materials. Due to their random lattice
of small cross sections and very high porosity, they
have great potential as carriers for cell immobiliza-
tion (Ogbonna
et al
. 1994). The sponges are strong,
chemically stable, and composed of interconnecting
voids within an open network of Fbers. Because of
their random lattice of small cross sections and high
porosity, the sponges are suitable for cell adhesion
(Phisalaphong
et al
. 2007).
This study aimed to evaluate the removal efFcien
-
cy of a bacterial consortium immobilized in a Fbrous
network of
Luffa cylindrica
on a mixture of methyl
parathion (MP) and coumaphos (COU) pesticides.
MATERIALS AND METHODS
Pesticides
To simulate a residue of OP pesticide mixtures,
two compounds that are authorized as OP pesti-
cides for agricultural and livestock use in Mexico
were selected, methyl parathion (O,O-dimethyl
O-4 nitrophenyl phosphorothioate)
and coumaphos
(O,O-Diethyl O-3-chloro-4-methyl-2-oxo-2H-chro-
men-7-yl phosphorothioate). Both pesticides were
purchased from Chemservice (99 % purity) (http://
web1.chemservice.com). Reactive grade ethyl acetate
was used as a solvent for pesticide extraction; HPLC
grade methanol from Mallinckrodt Baker Inc. (Phil
-
D.A. Moreno Medina
et al.
54
lipsburg, NJ, USA) was used to inject samples into
the gas chromatograph. All other chemicals were of
reagent grade and were obtained from J.T. Baker,
Mexico City.
Bacterial consortium
The bacterial consortium that was used in this
work was isolated from agricultural soils of the
Morelos state in central Mexico, which has a long
history of pesticide usage. Yáñez-Ocampo
et al
.
(2009) previously reported the characteristics of this
consortium.
Culture medium composition
To obtain bacterial biomass as a source of in-
oculum, soy tripticasein (ST), both agar and broth
methods were used (Bioxon, Becton Dickinson from
Mexico). For bacterial growth kinetics and pesticide
removal, we used a mineral salts medium (MSM) at
pH 7.00 ± 0.05; its composition per liter was 0.82 g of
K
2
HPO
4
, 0.19 g of KH
2
PO
4
, 0.20 g of MgSO
4
.7H
2
O,
2.00 g of KNO
3
and 0.99 g of (NH
4
)
2
SO
4
. Then, 2
mL of a trace solution containing 2.8 g/L H
3
BO
3
,
2.55 g/L MnSO
4
, H
2
O, 0.20 g/L CuSO
4
5 H
2
O, 2.43
g/L CoCl
2
6 H
2
O and 0.25 g/L ZnSO
4
7 H
2
O was
added to the MSM. In addition, glucose (0.1 %, w/v)
was added to the culture medium as a co-substrate
(Yáñez-Ocampo
et al.
2009).
Material used to support the immobilization of
cells
Loofa sponge, a natural material consisting of a
±brous network obtained from the matured dried fruit
of
Luffa cylindrica,
was used to immobilize bacterial
cells. The fruits of this Cucurbitaceae plant have a
±brous endocarp structure that is composed of cel
-
lulose (60 %), hemicellulose (30 %) and lignin (10 %)
(Mazali and Alves 2005). Loofa sponge is a good
support matrix for microbial cell immobilization
due to its high degree of porosity, high speci±c pore
volume, stable physical properties, biodegradability
and lack of toxicity for microorganisms (Liu
et al
.
1999, Iqbal and Saeed 2004, Nabizadeth
et al.
2008).
In addition, this material is low cost, which is another
advantage for developing countries. Because of these
excellent characteristics, its use as an immobilization
carrier is feasible.
Preparation of loofa sponge
Plant material (loofa sponge) obtained from the
matured dried fruit of
Luffa cylindrica
was used as an
immobilization carrier. Given that each loofa sponge
has a different structure, we selected sponges of simi-
lar size and structure,
i.e.,
similar in diameter, posi-
tion, size of the hollows and pore size of the ±brous
network. The native loofa sponge (about 70-80 cm
in length) has a complex structure, differing between
the core and peripheral regions. The sponge was cut
into appropriate segments for experimental use.
The fruits of
Luffa cylindrica
were dried to ob-
tain the ±brous network (FN). This procedure was
carried out in a dryer, at 70 ºC for approximately 4
days. Once the fruits were dry, the peel was removed,
and they were washed with distilled water and then
a solution of sodium lauryl sulfate (0.2 %) in order
to remove excess biomass. Washing was carried out
three times for 60 minutes at 60 ºC, and the FN was
dried again until it reached a constant weight. For
ease of handling, the sponge was cut into pieces of
approximately 1.0 x 1.5 × 0.4 cm and 0.1 cm thick.
The average weight of 100 pieces of sponge was
32.24 ± 6.89 mg. The sponge pieces were sterilized
at 120 ºC.
In addition, we used a second preparation method
for the sponge pieces. According to Ghali
et al
.
(2009) and Bal
et al
. (2004), the structural charac-
teristics of sponges improve when they are subjected
to pretreatment with NaOH. Therefore, NaOH 5 %
and anthraquinone were used as catalysts (Bal
et al
.
2004, Ghali
et al
. 2009). This treatment was carried
out at 80 ºC for 3 hours with constant stirring, and the
sponge pieces were subsequently washed three times
with deionized water. Finally, the pieces were dried at
70 ºC and stored in desiccators until further use (Iqbal
and Edyvean 2007). We performed a sterilization of
the sponge pieces prior to their use as supports.
Cells immobilization
For sponge colonization, the bacterial consortium
was inoculated into Erlenmeyer ²asks with 50 mL of
ST broth containing pieces of sponge and a mixture
of pesticides (1 %). To stimulate the formation of
bio±lm, the ²asks were incubated for 5 days at 28 ºC
and 100 rpm. Bacterial growth was measured by
optical density to reach the stationary phase of the
consortium. Then, the loofa pieces were washed
with sterile 0.5 % NaCl to remove the free cells.
The immobilized biomass in FN was used for further
kinetics.
Electron microscopy
To con±rm bacterial immobilization in the
Luffa
cylindrica
sponges, the pieces were collected and
treated using the Wu (2003) method (2.5 % glutaral-
dehyde treatment for 48-72 h, with repeated dehydra-
tions using 20 % acetone for a half-hour period, 50 %
REMOVAL OF PESTICIDES BY AN IMMOBILIZED BACTERIAL CONSORTIUM
55
acetone for 1 h and 100 % acetone for 7 hours at 4
o
C).
These samples were observed with a JSM 6400 JEOL
Scanning Electron Microscope (SEM) (
Fig. 1
).
Kinetics of bacterial growth and removal of the
mixture of pesticides with free cells
For removal experiments, three 125 mL sterile
Erlenmeyer fasks were supplemented with both
pesticides dissolved in Flter-sterilized methanol.
The methanol was evaporated to dryness, and then
the fasks were supplemented with 35 mL o± MSM
and 5×10
5
± 101 CFU/mL of the biomass. Previous
testing indicated that low bacterial activity degraded
the mixture of pesticides when supplied as a sole car-
bon source (data not shown); therefore, we decided
to measure the growth kinetics with the addition o±
glucose (0.1 %), in order to enhance pesticide bio-
degradation. The initial pesticide concentration was
25 mg/L and 5 mg/L for MP and COU, respectively.
MSM with pesticides and without the inoculum and
MSM with the inoculum and without pesticides
were used as controls. All fasks were incubated on
a shaking plat±orm ±or 72 hours at 125 rpm and 30
ºC. All treatments were performed in triplicate. Pes-
ticide removal and bacterial growth were measured
immediately after inoculation and several different
time intervals.
Adhesion of pesticides to the bacteria cells was
also tested. The consortium was cultured with ST
broth without pesticides for 24 hours; then the
culture was sterilized at 121
ºC for 35 min. There-
after, pesticides mixture (MP-COU) was added and
incubated for 7 days. Pesticides were then extracted
and quantiFed as described below (Ortiz-Hernández
et al
. 2003). Variations between pre- and post-in-
cubation of concentration of pesticides was 8.56 %
for MP and 7.34 % for MP and COU, respectively,
which was taken in consideration ±or the pesticide
removal.
Kinetics of bacterial growth and removal of the
pesticide mixture with immobilized cells in loofa
sponges
The removal of MP and COU by immobilized
cells in loofa sponges was analyzed. Pesticides
were provided as a principal carbon source as fol-
lows: three 125 mL sterile Erlenmeyer fasks were
supplemented with both pesticides dissolved in Flter-
sterilized methanol. The methanol was evaporated to
dryness, and then the fasks were supplemented with
35 mL of MSM, glucose (0.01 %) and immobilized
cells in loofa sponges, with and without sodium
hydroxide treatment, as described above. The initial
pesticide concentration was 25 mg/L and 5 mg/L for
MP and COU, respectively. In addition, two groups
o± Erlenmeyer fasks were used as controls: one was
inoculated with immobilized bacteria without pes-
ticides and the other with pesticides and loo±a Fber
without immobilized bacteria.
In order to quantify the growth of the immobilized
cells, the loo±a sponge bioFlm was Frst separated
to obtain free cells. The MSM was removed from
the loofa sponge, and a solution of 0.5 % NaCl was
added, and the mixture was stirred gently; then,
a phosphate buffer (0.4 M, pH 7) was added, and
the mixture was stirred gently for 2 minutes. This
procedure ensured that the immobilized bacteria in
the loofa sponge were sent into solution and that
they were removed from the loofa sponge for later
quantiFcation. ²ollowing this procedure, 1 mL was
taken to measure the number o± colonies by viable
count (CFU/mL). Separately, a scanning electron
microscope was used to verify the separation of the
bioFlm, and we ensured that all microorganisms pre
-
viously immobilized on loofa sponge were released
into the culture medium.
Analytical methods
²or pesticides quantiFcation, 1 mL aliquots were
collected ±rom each experimental fask and placed in
glass tubes. One milliliter of ethyl acetate was added
as an extracting agent, and the mixture was homog-
enized for three minutes using a vortex and allowed
to stand for two minutes. The organic phase was
recovered, and it was Fltered through a glass ±unnel
packed with glass Fber (Whatman G²/B) and anhy
-
drous sodium sulfate. The extract was collected in
amber vials; this procedure was repeated three times,
and 1 mL of ethyl acetate was mixed with the organic
phase each time. Finally, the content of the vials was
evaporated. These samples were reconstituted in 1
mL o± HPLC grade methanol ±or analysis (Yáñez-
Ocampo
et al
. 2009). Thus, extracted pesticides were
quantiFed on a gas Trace GC chromatograph coupled
to a Polaris Q Thermo Finnigan mass spectrometer
(GC-MS) using the EPA8141 method under the fol-
lowing conditions: equity column-5; 30 m × 0.25 mm
ID; 0.25 μm, oven at 120 ºC (3 min) and at 270 ºC
at 5 ºC /min, injector 250 ºC, MSD detector, scan
range 45-450 amu, 325 ºC trans±er line, helium fow
30 cm/s @ 120 ºC, injection 1.0 μL, splitless (0.3 min),
splitless liner, and double taper.
Statistical analysis
An analysis o± variance (α = 0.05) ±or each
variable (growth and degradation) was calculated.
D.A. Moreno Medina
et al.
56
The mean value of each treatment was analyzed by
multiple comparison of means, using Tukey’s test.
We performed a logarithmic transformation (Y =
log10X) for microbial growth data (CFU/mL). Data
were analyzed using the SAS version 9.0 (2002)
statistical package.
RESULTS
Treatment of
Luffa cylindrica
The average weight of 100 pieces of sponge
was 32.24 ± 6.84 mg, and the average weight of
the pieces after treatment was 24.58 ± 5.13 mg.
Therefore, a weight loss of 23.75 % was observed
after treatment. The morphological structure and
variability of the material treated with NaOH-AQ
were analyzed with a scanning electron microscope
(SEM). Observations suggest that the Fbers consist
of a relatively long central channel (lumen) with
small cells, 4 to 30 microns in diameter, which are
attached through a wall of approximately 2 to 3
microns in diameter, forming a complex network
within a growth axis of the fruit endocarp. Ghali
et al
. (2009) and Bal
et al
. (2004) reported that the
structural features and absorbency of the sponge
improve when it is subjected to a treatment with
sodium hydroxide (NaOH) (
Fig. 1
).
a)
b)
c)
d)
e)
f)
Fig 1.
Photomicrographs of
L. cylindrica
with immobilized cells (2000 X magniFcation).
Images from the left column belong to a loofa sponge without NaOH treatment.
Images from the right column were treated with NaOH. a) Inoculum and d) no
inoculum
REMOVAL OF PESTICIDES BY AN IMMOBILIZED BACTERIAL CONSORTIUM
57
Growth kinetics of cells in the suspension and
immobilized
The growth behavior of the bacterial consortium
in the suspension is shown in
fgure 2
. Growth ob-
served in the suspension was likely due to the com
-
bined action of glucose and the pesticide. To compare
consortium growth with the different treatments,
we carried out an analysis of variance including the
different treatments and the different culture times
during growth kinetics. Signifcant statistical diFFer
-
ences were Found only at 72 hours oF growth. Tukey´s
test revealed that the growth was enhanced when
pesticides were missing From the culture medium (P =
0.0074, α = 0.05). The controls did not show growth.
After releasing the FN microorganisms to the
culture medium, growth of the immobilized cells was
quantifed by the C±U/mL oF the culture.
Figure 3
shows the results of the bacterial consortium growth.
2.00E+06
1.80E+06
1.60E+06
1.40E+06
1.60E+06
CFU/mL
1.00E+06
8.00E+05
6.00E+05
4.00E+05
2.00E+05
0.00E+05
09
18
27
36
Time (hours)
MSM + Glucose
MSM + Glucose + Pesticides
MSM without consortium
45
54
63
72
Fig. 2
. Growth kinetics oF the bacterial consortium as Free cells. The controls did not grow during the
experiment
0.00E+00
2.00E+05
4.00E+05
6.00E+05
8.00E+05
1.00E+06
1.20E+06
1.40E+06
1.60E+06
1.80E+06
2.00E+06
0
9
18
27
36
45
54
63
72
81
CFU/mL
Time (hours)
Loofa sponge + MSM + consortium
Loofa sponge + MSM + consortium + pesticides
Loofa sponge
treated + MSM + consortium
Loofa sponge
treated + MSM + consortium + Pesticides
Fig. 3.
Growth kinetics oF the bacterial consortium immobilized on
Luffa cylindrica
. All cultures were
grown in MSM and 0.1 % glucose. Pesticides were tested at an initial concentration of 25 mg/L
and 5 mg/L of MP and COU, respectively. The bars on each line correspond to standard error
D.A. Moreno Medina
et al.
58
During the frst 48 hours, the number oF microorgan
-
isms found on the loofa sponge remained virtually
unchanged, but after that time, the microorganisms
began to grow, especially those immobilized in the
loofa sponge without pretreatment. The ANOVA in-
dicated that after 48 hours the growth was different
(P = 0.0001, α = 0.05), which was greater on untreated
fbers. This suggests that the consortium used may
more easily Form a bioflm on the fber with its origi
-
nal components. In addition, there was an increase
in growth when the pesticide was present. For FN,
regardless of treatment, the immobilized microor-
ganisms remained unchanged over time. However,
treatment with NaOH maintained the viability of
the bioflm For a longer period, independently oF the
presence or absence of the pesticide mixture.
Removal of pesticides mixture from culture
medium
Figure 4
shows the percentage of the MP-COU
mixture removed by the consortium in the suspen-
sion compared to the immobilized consortium. The
removal percentage in the suspension cultures, in the
case of MP, reached 55 %. However, we observed a
loss of 22 % in the control treatment, which may be
due to adhesion of pesticides to the bacteria cells,
abiotic losses or by errors in pesticide extraction prior
to quantifcation. In the case oF COU in the suspen
-
sion, removal was 62 % when bacteria were present,
with a loss of 15 % in the control treatment (without
bacterial consortium). However, pesticide removal
was better following treatment with the bacterial
consortium than without it. After 72 h, the pesticide
concentration reached 13.75 mg/L and 1.82 mg/L of
MP and COU, respectively.
Pesticide removal with the immobilized consor-
tium is shown in
fgure 4
. Due to the high removal
percentages, differences were observed when the FN
was not treated with NaOH. However, the results
show that these differences can be attributed to the
FN rather than the activity of bacteria. Most of the
MP and COU were not recovered from the culture
medium, regardless of the absence or presence of
microorganisms. These results suggest an adsorp-
Fig. 4
. Comparison of the removal percentages of pesticides following the appli-
cation of different treatments to the bacterial consortium. a) MP and b)
COU.
22.29
93
33.72
54.88
98.45
68.92
0
10
20
30
40
50
60
70
80
90
100
a)
b)
Free cells
Loofa sponge
without NaOH
Loofa sponge
with NaOH
%
Treatment
Control
Consortium
15
100
73.46
62
100
73.46
–20
0
20
40
60
80
100
Free cells
Loofa sponge
without NaOH
Loofa sponge
with NaOH
%
Treatment
Control
Consortium
REMOVAL OF PESTICIDES BY AN IMMOBILIZED BACTERIAL CONSORTIUM
59
tion process on the surface of the FN, which can
be very rapid. MP concentration was removed at
93 and 98 % in the control treatment and the im-
mobilized consortium, respectively, and COU was
removed entirely.
When the loofa sponge was treated with NaOH,
we observed signifcantly lower pesticide removal.
This was likely because the adsorption on the treated
FN was lower. The concentrations obtained at the
end of treatment were 10.07 and 1.25 mg/L for MP
and COU, respectively. The removal of MP follow-
ing treatment with the consortium was signifcantly
diFFerent (P = 0.0086, α = 0.05), while in the case
oF COU, the treatments did not diFFer signifcantly
(P = 0.277).
The abiotic losses of these pesticides may occur
due to adsorption by the support material, regardless
of the presence or absence of the inoculum, as previ-
ously reported for the adsorption of methylene blue,
crude oil and malachite green and even other xeno-
biotics (Annunciado
et al
. 2005, Demir
et al
. 2008,
Altinisik
et al
. 2010). We found that loofa sponges
without pretreatment provided increased removal of
both MP and COU.
Overall assessment of pesticide removal by the
consortium
AFter measuring growth and removal kinetics,
an overall comparison of all the treatments was per-
formed. For the statistical analysis, three variables
were included: microbial growth, removal of MP and
removal of COU. We used an analysis of variance,
and when the results were statistically signifcant,
Tukey’s test was applied.
For bacterial growth, we performed a logarith-
mic transformation of the data (Log10X, where
X = number oF C±U/mL). The ANOVA results oF the
last values oF the growth kinetics revealed signifcant
differences in the growth of the bacterial consortium
in response to the diFFerent treatments (P = 0.0057,
α = 0.05). We observed that cells in the suspension
showed the best growth, and there were no signifcant
differences in the presence or absence of the pesticide
(
Table I
). However, it is important to note that the
cultures with immobilized cells showed prolonged
viability, which could be advantageous for practical
applications of remediation.
For the statistical analysis of MP and COU
removal (%), arcsine transformation of data was
perFormed (arcsin √ X, where X = percentage oF
pesticide removal). The ANOVA results revealed
significant differences between the treatments
(P = 0.0002, α = 0.05) (
Table I
).
TABLE I.
COMPARISON OF THE CONSORTIUM GROWTH AND PESTICIDE REMOVAL UNDER DIFFERENT TREATMENTS. THE CULTURE MEDIUM
CONSISTED OF MSM AND GLUCOSE (0.1 %). THE DATA IN TABLE WERE CALCULATED FROM TRANSFORMING GROWTH (FCU / ML) AND
REMOVAL (%), AS MENTIONED PREVIOUSLY
Treatment
Growth
(CFU/mL)
Tukey
(Growth)*
MP removal
(%)
Tukey
(MP)*
COU
removal (%)
Tukey
(COU)
With consortium
Free cells + pesticides
6.15
a
0.68
c
0.68
c
Free cells without pesticides
6.24
a
-
-
-
-
Immobilized in a loofa sponge without NaOH treatment + pesticides
5.55
ab
1.45
a
1.44
a
Immobilized consortium in a loofa sponge without NaOH treatment
without pesticides
5.29
b
-
-
-
-
Immobilized in a loofa sponge with NaOH treatment + pesticides
5.24
b
0.85
bc
0.85
bc
Immobilized consortium in a loofa sponge with NaOH treatment without
pesticides
5.40
ab
-
-
-
-
Without
consortium
MSM + pesticides
0.00
c
0.44
c
0.44
c
Loofa sponge without NaOH treatment + pesticides
0.00
c
1.30
ab
1.30
ab
Loofa sponge whit NaOH treatment + pesticides
0.00
c
0.62
c
0.62
c
*DiFFerent letters indicate signifcant diFFerences (P <0.05)
D.A. Moreno Medina
et al.
60
The results of this analysis confirm the data
mentioned above, and we concluded the following:
The growth of bacterial consortium was higher
in suspension and only showed statistical differ-
ences after 72 hours of culture, being higher when
pesticides were absent.
In accordance to Tukey´s test, after 48 hours the
growth of immobilized consortia was signiFcantly
greater on Fber loofa sponge without pretreatment.
The treatment with NaOH maintained the viability of
the bioFlm for a longer period, independently of the
presence or absence of the pesticide mixture.
MP removal is more efFcient when using the ±N
whether in presence or absence of microorganisms,
which suggests a strong and fast adsorption on
Loffa
cylindrica
Fbers.
Similar behavior was observed for COU, although
differences were found when the consortium was
present, indicating a lower adsorption as a result of
treatment and higher microorganism activity.
DISCUSSION
The enzymatic degradation of organophospho-
rus pesticides as a treatment strategy for their Fnal
disposal has gained attention over the last 20 years,
because it has advantages over other treatments;
namely, it does not generate additional waste, and
it is relatively cheaper than physical and chemicals
treatments. However, it remains important to develop
strategies for its implementation, such as identifying
low cost solutions, especially in developing coun-
tries. Here, we attempted to optimize a system for
degrading a mixture of pesticides that are generated
as a waste at the end of their use. We developed a
simple procedure for the immobilization of bacteria
by adsorption on the surface of the Fbrous network
of
Luffa cylindrica
, which is inexpensive and readily
available in central Mexico.
Microorganisms can be immobilized in differ-
ent matrices, whether the cells are entrapped in the
support material or an adsorption phenomenon oc-
curs. Immobilized microbial cells have mainly been
used to produce useful chemicals and for xenobiotic
degradation. The material used for immobilization
must be inert, non-toxic to cells, and preferably
economical and practical. The main advantages
of using biomass immobilization techniques are;
the high cell density achieved, the reuse of cells is
enabled, high operational stability and an increased
rate of degradation of the substance being processed,
also, the washing of biomass cells is avoided, which
is advantageous in continuous processes (Kadakol
et al
. 2011).
Yáñez-Ocampo
et al
. (2011) and Yáñez-Ocampo
et al
. (2009) studied the use of a consortium of bacte-
ria immobilized in volcanic rock as an alternative for
the degradation of organophosphorus pesticides, and
they found that this method had a high rate of MP re-
moval (66 and 70 %). In this study, we removed 100 %
of the MP and COU added to the culture medium.
Various studies have demonstrated that bioFlms
formed on substrates, used for immobilization of
microorganisms, are less sensitive to environmental
changes, such as temperature and pH, compared with
bacteria cultured in suspension and in presence of
metabolic products and toxic substances (Ohandja
and Stuckey 2006). Importantly, we found that the
highest removal was obtained by the activity of the
Fbers, rather than the activity of microorganisms.
This is likely because the adsorption is very fast and
strong.
Experiments testing the removal of MP with
immobilized cells indicated that the sponge Fbers
contribute to the removal of the pesticide from cul-
ture media. Thus, abiotic losses of this pesticide can
be attributed to adsorption into the support material
and adhesion to the bacterial cell. These results sug-
gest that MP and COU are removed from the media
mainly by adsorption and not by degradation, since
a removal in the control (culture media with Fber
sponge without bacteria) could be observed. Further-
more, pesticide degradation experiments revealed
that the removal of COU was more complete than
MP. This could be due to the characteristics of the
pesticide and/or the different concentrations used in
the assay.
Pattanasupong
et al
. (2004) reported the use of F
-
ber
Luffa
sp. as a support material for immobilization
of a bacterial consortium to simultaneously degrade
two pesticides, the fungicide carbendazim and the
herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D).
Similar results were obtained after treatment with
bioFlm immobilized on
Luffa
sp., without pretreat-
ment, which presented the highest values of MP and
COU removal.
The present study suggests that it is possible to
increase the efFciency of MP and COU degradation
using cells immobilized on a loofa sponge compared
with free cells in a suspension. Thus, immobilized
microbial technology is a very versatile approach
that could be used for the degradation of toxic pol-
lutants from industrial ef²uents. Moreover, the loo
-
fa-immobilized cells systems have been efFciently
used for the treatment of wastewaters containing
REMOVAL OF PESTICIDES BY AN IMMOBILIZED BACTERIAL CONSORTIUM
61
toxic metals, dyes and chlorinated compounds, and
the technology has been used to develop bioflms For
the remediation of domestic and industrial waste-
waters rich in inorganic and organic matter (Saeed
and Iqbal 2013).
Further studies are needed to thoroughly elucidate
the mechanisms by which pesticides are adsorbed on
±N, whether the adsorbed pesticides are likely to be
degraded or whether they stay in the FN. Only until
then it will be possible to recommend potential ap-
plications For the degradation oF pesticides in specifc
locations.
CONCLUSIONS
In order to degrade pesticides, it is important to
search for materials with favorable characteristics for
the immobilization of cells, including aspects such as
physical structure, ease of sterilization, the possibility
of repeated use, and affordable.
In this study, the microbial consortium immobi-
lized on loofa sponges exhibited a high ability to re-
move both MP and COU. The use of an immobilized
consortium is thought to be more advantageous than
the use of free-living consortium when applied for
the bioremediation of water contaminated by these
pesticides. Loofa sponges are considered one of the
most suitable supports for this application, since
they are renewable, biodegradable, and affordable
(and thus viable for use in developing countries),
in addition, the microbial immobilization method
is simple. Although the degradative ability of this
consortium was not distinguished by the adsorption
that occurred in the FN and to the bacteria cells,
the system developed could be applied to degrade
pesticides and develop further studies to ensure its
subsequent desorption and degradation. Information
about the effect of various environmental factors on
the degradation of MP and COU would facilitate its
application.
ACKNOWLEDGEMENTS
We would like to thank Dr. Andrés Aguilar
Negrete from the Instituto de Ciencias Físicas
(UNAM) for his collaboration with SEM micro-
graphs. We also thank Dr. Rosa Cerros Tlatilpa From
Laboratorio de Sistemática, Facultad de Ciencias
Biológicas, UAEM, For taxonomic identifcation
of
Luffa cylindrica
.
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